Field of the Subject Disclosure
The present subject disclosure relates to imaging for medical diagnosis. More particularly, the present subject disclosure relates to identifying physiologically plausible stain vectors within an assay.
Background of the Subject Disclosure
In the analysis of biological specimens such as tissue sections, blood, cell cultures and the like, biological specimens are stained with one or more fluorophores or chromogens, and subsequently scanned or photographed for analysis. Observing the signals generated from the scan enables a variety of processes, including diagnosis of disease, assessment of response to treatment, and development of new drugs to fight disease. An assay includes biological specimens such as tissue sections from human subjects that are treated with a stain containing a fluorophore or chromogen conjugated to an antibody which binds to protein, protein fragments, or other targets in the specimen. Upon scanning the assay, multiple channels of image data including color channels are derived, with each observed channel comprising a mixture of multiple signals.
Generally, color separation (or spectral unmixing) is used to determine a concentration of specific stains within an observed channel or channels of an assay. This may also be known as color de-convolution. Each pixel of a scanned image is represented by a vector of image values, or a color vector, and each stain corresponds to a reference vector, also known as a reference spectrum. The local concentration of the stain is represented by a scaling factor of a reference vector. Therefore, the color vector for a pixel that contains multiple co-located stains with different concentrations is a linear combination of the reference spectra of all the present stains. Typically, fluorescence imaging color channels directly provide the image vector and reference spectra. In brightfield (transmission) imaging, light intensities emitted by the stained tissue are transformed into an optical density space, with mixing of different stains being represented by a linear weighted combination of the contributing reference spectra.
The unmixing process extracts stain-specific channels to determine local concentrations of individual stains using reference spectra that are well-known for standard types of tissue and stain combinations. However, the reference spectra for pure stains tend to vary with tissue type, controlled and uncontrolled process parameters during staining, and with age. For instance, there are always variations within a tissue type based on age of the tissue, age of the stain, how the tissue was stored, dehydrated, fixed, embedded, cut, etc. These variations can influence how a stain will appear, and can result in unwanted artifacts in the results of an unmixing process. Existing methods cannot handle such errors without human guidance, and no reliable reference spectra are available for such variations. Therefore, incorrect separation and physiologically or physically implausible results continue to occur. Moreover, with respect to bright field images containing 3 color channels, any co-location of greater than 3 stains cannot be unmixed, or no unambiguous mathematical solution exists.